title: GR_ChIP-seq_dex_washout
description: Protocols: ChIP-seq was performed on 30 million cells. Cells were cross-linked with 1% formaldehyde for 3 minutes and harvested. Cells where grown to approximately 90% confluence according to standard protocols in DMEM medium with 5% FBS. Cells were treated with 100 nM dexamethasone for 4 hours, washed 2x with PBS and cultured for 24 hours in hormone-free medium before harvest. Cells were lysed with IP lysis buffer (50 mM HEPES-KOH pH 7.4, 1mM EDTA, 150 mM NaCl, 10% Glycerol, 0.5 % Triton X-100, 1:200 Protease inhibitor, 1:200 PMSF). After centrifugation pellets were resuspended in RIPA buffer (10 mM Tris-HCL, pH 8, 1 mM EDTA, 150 mM NaCl, 5 % Glycerol, 0.1 % DOC, 0.1 % SDS, 1 % Triton-X100, 1:200 Protease inhibitor, 1:200 PMSF) and sonicated with Diagenode Bioruptur using 24 cycles (30 sec on, 30 sec off) at 4 °C. After centrifugation lysate was resuspended in RIPA buffer. The antibody was added and incubated overnight. A 50% agarose bead slurry (Santa Cruz protein A/G agarose beads) was washed 2x with RIPA buffer and nutated overnight at 4 °C. RIPA buffer was removed and beads were added to the samples and incubated rotating for 4h at 4 °C. The beads were washed 5x with 1 ml RIPA wash buffer (RIPA containing 500 mM NaCl). ChIP samples were supplemented with Proteinase K solution (TE buffer pH 8, 0.7 % SDS, 200 µg/ml proteinase K) and incubated for at 55 °C for 3 h and 65 °C overnight. Next day 1 µl RnaseA (2 µg/ml) was added and incubated at 25 °C for 1 h. The ChIP samples were purified using the PCR Purification Kit (Promega, Wizard SVGel and PCR Clean-Up system). Antibody used: GR, N499 (2 µl/5 million cells) Sequencing libraries were prepared using the Kappa HyperPrep Kit (Roche 07962363001) according to manufacturer's instructions.
attributes.tag: ENA first public
attributes.value: 2021-05-12
attributes.tag: ENA last update
attributes.value: 2021-05-12
attributes.tag: External Id
attributes.value: SAMEA7722233
attributes.tag: INSDC center name
attributes.value: MPIMG
attributes.tag: INSDC first public
attributes.value: 2021-05-03T00:22:16Z
attributes.tag: INSDC last update
attributes.value: 2020-12-15T10:39:43Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9914:GR_ChIP-seq_dex_washout
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: U2OS-GR
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: osteosarcoma
attributes.tag: organism part
attributes.value: bone
attributes.tag: sample name
attributes.value: E-MTAB-9914:GR_ChIP-seq_dex_washout
attributes.tag: stimulus
attributes.value: compound and washout